Role of histidine-50, glutamic acid-96 and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin

Lacadena García-Gallo, Francisco Javier; Martínez Del Pozo, Álvaro; Martínez Ruiz, Antonio; Pérez-Cañadillas, José Manuel; Bruix, Marta; Mancheño Gómez, José Miguel; Gavilanes Franco, José Gregorio; Oñaderra Sánchez, Mercedes

Role of histidine-50, glutamic acid-96 and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin

Download

10 - 1999_Proteins_Mutantes.pdf (425.37 KB)

Publication date

1999

Publisher

Wiley

Citations

Exportar

URI

https://hdl.handle.net/20.500.14352/108286

Citation

Lacadena, J., Martínez del Pozo, Á., Martínez-Ruiz, A., Pérez-Cañadillas, J.M., Bruix, M., Mancheño, J.M., Oñaderra, M. and Gavilanes, J.G. (1999), Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin α-sarcin. Proteins, 37: 474-484. https://doi.org/10.1002/(SICI)1097-0134(19991115)37:3<474::AID-PROT14>3.0.CO;2-N

Abstract

alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.